Multidrug-resistant tuberculosis (MDR-TB) remains a public health emergency and a threat globally. Although increasing MDR-TB cases have been recently reported in Somalia, limited information is known. This study aims to determine the prevalence of drug-susceptible and MDR-TB in suspected patients referred to the TB Department in Mudug Hospital, Galkayo, Somalia, and identify potential factors associated with MDR-TB. A 3-year hospital laboratory-based retrospective study was conducted by manually reviewing laboratory records of Mycobacterium tuberculosis specimens and GeneXpert MTB/RIF results from January 2019 to December 2021 at the reference mycobacteria laboratory department in Mudug Hospital. A total of 714 positive GeneXpert-MTB results were identified: 619 (86.7%) were drug susceptible (no Rifampin resistance [RR] detected) and 95 (13.3%) with RR detected or defined as MDR-TB. Most of the MDR-TB patients were males (71.6%, 68/95) and between the ages of 15 to 24 (31.6%, 30/95). Most isolates were collected in 2021 (43.2%, 41/95). Multivariate analyses show no significant difference between patients having MDR-TB and/or drug-susceptible TB for all variables. This study showed an alarming frequency of MDR-TB cases among M. tuberculosis-positive patients at a regional TB reference laboratory in central Somalia.